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Expression and function of globin X in nucleated red blood cells of fish

Final Report Summary - GBX IN FISH RBCS (Expression and function of globin X in nucleated red blood cells of fish)

4.1 Final publishable summary report

The aim of the project is to understand the function of globin X (GbX) in the activation of the sodium/proton exchange across the erythrocyte membrane of fish. The sodium/proton exchange is oxygen dependent and activated by adrenergic stimulation; however the exact mechanism is unknown. It has been suggested that membrane binding of haemoglobin would be involved but such haemoglobin has not been found yet. Recently we found that GbX mRNA is expressed in red blood cells of fish. GbX is a membrane-bound vertebrate globin that is unique within the vertebrate (haemo-) globin superfamily and its cellular functions are not well understood. In a multidisciplinary approach we combined the molecular and functional findings on GbX and oxygen-dependent membrane transport in a completely new fashion with an environmental focus. The study included in vitro approaches in red blood cell culture and in vivo experiments in different fish species.

Work performed and main results
The first objective aimed at identifying conditions that lead to an increase in GbX transcripts and/ or protein content in fish RBCs. We used three different salmonid fish species, which differ in their temperature and oxygen preferences: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Arctic char (Salvelinus alpinus) for our in vivo and in vitro experiments.
In the in vitro studies in rainbow trout red blood cell (RBCs) we found that transcript levels of GbX were either unaffected or lower after most treatments (hypoxia, hyperoxia, temperature increases and β-adrenergic stimulation) compared to control samples. Since cellular steady-state mRNA levels are determined by the transcription rate and the degradation of the transcript, we then investigated how hypoxia and β-adrenergic stimulation affected the transcription rate and stability of various transcripts of interest. Originally we planned to use the nuclear run on method (NRO) described by Sambrook & Russell 2001 (Molecular Cloning A, Cold Spring Harbor Laboratory Press, NY) to measure transcription rate, but during the experiments it turned out that the method, which was optimized for the use in mammalian cells, is not well suited for fish. This was an unexpected problem and the optimization of the protocol took longer than expected. The revised version of the protocol was then successfully applied to three research questions in different fish cell types (RBCs, liver cells, and a gill cell line). In rainbow trout RBCs and the gill cell line the transcription rate of GbX could not be determined because of only sporadic amplifications which are probably a result of very low transcription rates. The stability of the GbX transcript was compared to other transcripts low and thus the half-life short. None of the transcripts included in the study changed its rate in response to the treatments in RBCs. However, the stability of two transcripts was significantly affected and other transcripts showed a tendency for longer half-lives (e.g. haemoglobin, beta-actin). These results have important implications for many studies because often changes in the steady-state levels are used as indicators for changes in transcription rate of the respective transcripts.

In two in vivo experiments we then evaluated the changes in the transcriptome of RBCs and spleen after long-term exposure to hypoxia (12 h to 24 h) and reoxygenation. The fish spleen is an important RBC reservoir and during periods of increased oxygen demand, it releases up to 95% of the stored RBCs into the general circulation. The experiments were done in collaboration (University of British Columbia, Canada; Natural Resources Institute Finland (Luke), Finland) and aimed at understanding the expression of globin genes and other candidate genes in the red blood cells (RBCs) and the spleen of salmonids during hypoxia and reoxygenation after temperature acclimation. Fish were acclimated to two different temperatures (8°C and 15°C) for three weeks and then exposed to hypoxia for up to 12 hours followed by a reoxygenation phase of 12 hours. The first results show that hyperoxia and short-term hypoxia (6 h) left the transcript levels largely unaffected. Only long-term hypoxia increased the transcript levels of GbX. Part of the samples were subjected to RNAseq and the analyses are still ongoing. To sum up the results obtained so far in the first part, we found that the expression of GbX and its regulation showed high variability among individuals, and that the factors controlling the expression are still not clear. The proof of the GbX protein in RBCs by mass spectrometry was impossible because of the very low expression.
The N- and C-terminal extensions of GbX are unique and their functions are largely unknown. We designed several constructs of the rainbow trout GbX CDS to evaluate the effect of each part on the cellular localization using confocal microscopy. The rainbow trout GbX has the same targets for dual fatty acid acylation (myristoylation, palmitoylation) which are characteristic for this protein. These lipid modifications localize the protein to the cellular membranes. In cell culture we could show that the N-terminal motif is the only determinant of the localization and it will target the protein to the inner and outer membranes independent of the rest of the protein. In a collaborative study we evaluated the dynamics of myristoylation of the GbX protein of rainbow trout and zebrafish. The work is close to completion and the manuscript is in preparation.
The objective to identify the protein interactome of GbX.was very ambitious and, unfortunately, could not be accomplished during the two-year duration of the project. As planned, we prepared the plasmid constructs (V5- and His-tagged) and successfully transfected cells with it, but we were not able to capture the expressed protein with magnetic beats. Trials with a different approach have been started already and are still ongoing.

In conclusion, the project yielded a large amount of novel data especially on gene expression regulation in fish. Currently there is no evidence that GbX expression is directly regulated by β-adrenergic stimulation. Some analyses are still ongoing because of time constraints caused by the time-consuming optimization of the NRO method already in the beginning of the project.

In summary, from the project results five scientific papers are either already submitted or are in preparation. In addition, parts of the results have been presented in three international conferences. One PhD thesis directly profited from the development of a revised protocol of the NRO method to measure transcription rate in fish cells, and the fellow was officially appointed as a supervisor for the PhD student. Moreover, a Master’s student and several students of Finnish and international laboratory technician schools were involved in the project. Together with a local company the applicant tested and developed further a microplate reader system with broad applications in life science and medical research, and an application note was published together with the company. During the course of the fellowship a number of collaborations were started. Furthermore, the fellow has strongly developed and matured as a researcher, acquiring both transferable and management skills.